Sequencing phenomenon

>Andrea M Skantar (skantar at unity.ncsu.edu) wrote:
>: Perhaps this has been covered before, if so, pardon the repetition. Does
>: anyone know what causes a wierd phenomenon in sequencing gels where the
>: tracking dye in the center goes SLOWER than on the edges? I've heard
>: about the dye going slower at the edges and faster in the center being
>: due to non-uniform heating of the gel, but this is a new one on me. It
>: gives the gel like a fish-eye effect at the bottom. I've gotten readable
>: sequence from such gels but it sure looks wierd. Any ideas?
>: Andrea
>> Have you heard of wedge gels? Wedge gels use a spacer that is
>thicker at the bottom than at the top, so those small DNA fragments that
>would otherwise run off the bottom of a normal gel after a long run slow
>down and will be retained on the gel. This happens because there is less
>current per cross-sectional area flowing through the thick part of the gel.
>This could account for the center of a normal gel migrating slower than
>the edges. If you pour a gel and polymerize it in a vertical or slanted
>position the plates will bow out ever so slightly to make the center of
>the gel thicker than the edges.
> Glass plates are not as rigid as one would think. They are
>actually quite easy to bend. To get a perfectly uniform gel you need to
>lay it nearly flat during polymerization.
>>--
>* Brian Foley * If we knew what we were doing *
>* Molecular Genetics Dept. * it wouldn't be called research *
>* University of Vermont * *
This may or may not be the reason for Andrea's problem, but I've had the center
of the gel run slower (i.e., the dye front is a frown) if I get my plate
orientation mixed up. What happens is that sometimes, all the marks get washed
off my plates (for instance, if I clean them with base) and when I pour my next
gel and run it, the dye front frowns and all the bands follow that pattern on
the autorad. I have often noticed that when I pry the plates apart after
running the gel that the gel itself is VERY thin in the lower middle of the gel.
My reasoning for what's happened is (as Brian mentioned above) that the plates
have warped due to repeated use in one position and now I've suddenly put them
in another position and they aren't really flat anymore. My suggestion is to
try some different plate orientations if you think this might have happened to
you Andrea.
Estelle Hrabak>